【作者】 张旭华；

【导师】 王传新；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2012， 博士

【摘要】 目的：结直肠癌在世界范围内死亡率排名第三,而结直肠癌患者的生存率与患者发现时的肿瘤分期密切相关,发现越早生存率越高。结肠镜大大提高了结直肠癌的诊断率,但是它有创、昂贵及不方便操作等特点限制了它的应用。而血清肿瘤标志物以其无创经济快速等特点成为了全世界相关研究人员关注的对象。目前结直肠癌的肿瘤标志中最为可靠并广泛应用的只有CEA,这远远不能满足临床需求,本研旨在用定量蛋白质组学技术寻找能够作为早期诊断或预后判断的新的肿瘤标志物。对候选肿瘤标志物ORM2分析其在结直肠癌组织中的表达,探讨其在结直肠癌组织分级和临床分期中的表达差异；检测ORM2在结直肠癌及良性肠道疾病患者血清中的含量,探讨血清ORM2水平在良恶性肠道疾病鉴别诊断中的价值。方法：1)用定量蛋白质组学方法ITRAQ分析。收集结直肠癌病人血清10例与正常对照组血清10例,提蛋白后去除12种高峰度蛋白,然后用trypsin进行蛋白消化得到肽段。用iTRAQ试剂标记消化后的肽段,114标记癌症病人组,117标记正常对照组。将标记的肽段混合后运用强阳离子交换柱(strong cation exchange, SCX)和C18柱进行分离,最后运用MALDI-TOF/TOF和micro-QTOF质谱进行鉴定,得到差异蛋白。2)通过MetaCore生物网络分析,分析差异蛋白,预测差异蛋白的在生物网络中的关系。3)采用western blot法确认ORM2蛋白在41例结直肠癌组织及41例癌旁正常结直肠组织中的表达,说明ORM2的表达在癌组织与正常组织间的不同。4)应用RT-PCR的方法对结直肠癌患者术后病理组织标本及相应的结直肠癌瘤旁正常肠组织进行mRNA水平的验证。5)采用ELISA双抗夹心法和化学发光法分别检测了180例结直肠癌、59例结直肠息肉、53例炎性肠道疾病、62例腺瘤和65例正常健康组的血清ORM2和CEA水平,并计算其统计学意义。6)应用受试者工作特征曲线(Receiver operating characteristic curve, ROC)分别评价ORM2和CEA在鉴别诊断良恶性肠道疾病中的敏感性及特异性。结果：1)用ITRAQ定量蛋白质组学方法鉴定差异蛋白,最终共发现76个差异蛋白点,其中两个或两个以上肽段进行定量鉴定的蛋白有61个,采用单个肽段进行定量鉴定的蛋白有15个。分别用DATA ANALYSIS4.0软件与WARP-LC软件分析质谱数据,MASCOT搜索质谱匹配蛋白,在鉴定出的76个蛋白点中,以正常组为对照,其中有13个差异表达蛋白具有统计学意义,其中9个表达上调,4个表达下调,这13个差异蛋白的功能涉及到补体系统、糖蛋白等。2)将得到的13种差异蛋白上传MetaCore进行在线分析。得到ORM、补体系统等相互作用的网络图,在网络图中每一个蛋白占据一个节点,利用节点蛋白表达的上调下调信息,还可以推断可能变化的蛋白质。3) Western Blot结果显示癌组织中ORM2蛋白表达为正常组织的2.55倍(P<0.01),具有显著性差异,与蛋白质组学结果一致。(p<0.01)。4) RT-PCR结果显示癌组织中ORM2基因表达为正常组织的2.65倍(P<0.01).5)血清ORM2的浓度在结直肠癌组的浓度明显高于正常对照组、结直肠息肉组和腺瘤组(P<0.001)。血清ORM2浓度在肠道炎症性疾病中的浓度明显高于正常对照组、结直肠息肉组和腺瘤组(P<0.001)。肠道炎症性疾病病人的血清ORM2浓度明显高于结直肠癌症组(p<0.05);结直肠癌组血清ORM2浓度和年龄、性别、组织类型和TNM分期物明显差异(p>0.05)。血清CEA升高和正常组的CRC病人之间血清ORM2水平差异显著(p<0.05)6)ORM2的ROC曲线面积为0.676,Cutoff为176.9U/mL,此时,其诊断结直肠癌的敏感度67.8%,特异度为62.8%结论本实验首次将定量蛋白质组学ITRAQ技术应用于结直肠癌肿瘤标志物的研究当中。ITRAQ技术在肿瘤标志物发现方面可以与传统蛋白质组学呼应,具有较高的重复性和经确性,能够发现一些候选的标志物。对筛选出的候选肿瘤标志物ORM2的验证得出,结直肠癌组织中ORM2在蛋白水平和ImRNA水平表达均明显升高,血清ORM2水平在结直肠癌患者中显著升高,同时肠道炎症性疾病血清ORM2明显增高。这就说明血清ORM2在结直肠癌的发生发展过程中有一定的作用,尤其是在肠道炎症性疾病进展而成的结直肠癌病人当中,或许有一定的作用,同时也有可能成为结直肠癌诊断或预后判断的标志物,通过ROC曲线评估ORM2在鉴别诊断结直肠癌患者和良性肠道疾病方面具有较高的敏感度及特异度,在敏感性上面具有相对优势,但在特异度方面欠缺。ORM2或许会成为肠道炎症性疾病到结直肠癌演变过程当中的一个标志物。更多还原

【Abstract】 ObjectiveColorectal cancer (CRC) is the third most common malignancy in the world. The risk of death is closely correlated to the stage of CRC at the time of primary diagnosis. Sigmoidoscopy significantly improved the detection rate of CRC. However, its wide application is still limited due to high costs and inconvenience. Serum tumor marker which can be easily quantitatively measured, reproducible and cost effective became the hot subjects throughout the world. Plasma/serum markers Carcinoembryonic antigen (CEA) are the only widely used and reliable tumor markers for CRC. That is far from the clinical needs. Thus, specific sera-based tumor markers for early diagnosis and prognosis are needed. Therefore, the purpose of this study is to identify serum biomarkers of CRC. In additon, we further evaluated the potential role of the candidate biomarker ORM2in the differential diagnosis of colorectal cancer and benign colorectal diseases.Method1) We used a quantitative proteomic approach with isobaric labeling (iTRAQ) to examine changes in the plasma proteome of10patients with CRC compared to healthy volunteers. After depleting of12abundant proteins the samples were digested with trypsin and labeled with iTRAQ reagents (114for the CRC group,117for the healthy group). And then MS analysis was performed with MALDI TOF/TOF MS and MICROQ-TOF MS. The differential expressed proteins were identified after DATABASE research. 2) The software MetaCore was used to analyze the relationship of the candidate proteins in the biological network.3) Western blot method was used to confirm the expression of ORM2in CRC patients.41specimens of patient with CRC and41specimens form non-tumorous controls were detected by Western blot to confirm the expression of ORM2from normal controls.4) RT-PCR was performed to validate the differential expression of the candidate between carcinoma and normal tissues on the level of mRNA.5) Enzyme-Linked Immunosorbnent Assay (ELISA) was performed to measure ORM2in serum from419subjects:normal control (n=65), hyperplastic polyp (n=59), inflammatory bowel disease (IBD)(n=62), adenoma (n=53) and CRC (n=180). The CEA was detected using the chemiluminescent microparticle immunoassay technology. Kruskal-Wallis test was applied to determine statistically significant differences, P<0.05was considered as statistically significant. Receiver operating characteristic (ROC) curve was used to evaluate the sensitivity and specificity for ORM2and CEA. All statistical analysis and receiver-operator characteristic (ROC) curve were performed with SPSS software versions13.0for windows (SPSS Inc, Chicago,Ill).Result1) In our quantitative proteomics analysis, we detected76human plasma proteins with more than95%confidence using iTRAQ labeling in conjunction with micro Q-TOF MS. Among this group,13differentially expressed proteins were selected based on1.5-fold over-expression and1.5-fold under-expression in CRC patients, compared to the healthy volunteers.9up-regulated and4down-regulated proteins were observed in the CRC group. The statistical variance of tumor versus normal ratios was within the95th confidence level (P=0.05).2) The network analysis of the differential proteins revealed ORM, complement system and RBP4involvement in CRC.3) When protein expression was measured by densitometer, the median densitometer value of ORM2in CRC cancer tissues was significantly greater than that in corresponding adjacent normal mucous tissues (P<0.01)4) RT-PCR showed that expression of ORM2in CRC was2.65(P<0.01) higher than in normal tissues.5) Median plasma ORM2concentrations were significantly higher in CRC compared to the normal colorectum, hyperplastic polyp, and adenoma (all at P<0.001, respectively). The plasma ORM2level was statistically significantly higher in patients with IBD than in the normal colorectum, colorectal hyperplastic polyp and adenoma (all at P<0.001, respectively) and it was statistically significantly higher in patients with IBD than in CRC (P<0.05). No significant association was found between plasma ORM2concentrations and age, gender, TNM stage or histological grade.Conclusion:Our study for the first time coupled iTRAQ with Q-TOF/MS on plasma specimens of CRC patients to discover potential biomarkers for CRC.13different expressed proteins were identified. Most of them have not been reported as cancer biomarkers that have shown iTRAQ together with Q-TOF/MS is a sensitive, reproducible, robust technique for identifying statistically significant differences of protein expression between the CRC patients and healthy control plasma samples. From the verification of ORM2, it was found that ORM2may be a potential biomarker to distinguish CRC from IBD. In future studies, larger sample populations will be required to confirm these potential biomarkers. In our research, ORM2is firstly identified as a differently regulated protein in CRC patients as compared to normal controls with proteomics screening. However, it requires a lot of bench experiments and clinical tests before ORM2can be used as a reliable biomarker.更多还原